Studies on DNA replication of plasmid Co1E1 have been continued. Transcripts (RNA II) that start 555 nucleotides upstream of the replication origin by RNA polymerase form a hybrid with the template DNA. The hybridized transcript is cleaved by ribonuclease H at the origin and used as the primer for DNA synthesis by DNA polymerase I. Primer formation is affected by point mutations. Each of them affect a specific stage of primer formation and inactivated primer. Studies on RNA II structure by a computer program under constraints which are based on biochemical and genetic data show that functional RNA II has a unique secondary structure that fold in a specific tertiary conformation. Primer formation is regulated by a plasmid-specified small RNA (RNA I). RNA I binds to RNA II at the complementary region. This binding results in inhibition of formation of the secondary structure necessary for primer formation. Binding starts by reversible interaction between loops of folded structures. This interaction facilitates stable binding that initiates at the 5'-end of RNA I. Primer formation is also regulated by a 63-amino acid protein (Rom) specified by the plasmid. The protein stimulates binding of RNA I to RNA II by affecting initial reversible interaction of these RNAs. Thus the protein enhances the inhibitory action of RNA I. Inhibition of primer formation by RNA I in the presence or absence of the protein determines the copy number of a plasmid in a cell and the incompatibility between related plasmids.